Conjugation of antigens to molecules that bind receptors expressed on dendritic cells (DC) has been shown to increase immunogenicity in several ways: 1) DC targeting increases Ag delivery to DC 100-1000 fold; 2) targeting receptors that activate DC provides co-stimulatory signals that mimic the effects of traditional non-specific adjuvants; 3) because such receptors are pathogenic-specific, their activation results in immune responses that mimic that of natural infection. Although proven in concept, a practical method of targeting antigens to DC-activating receptors does not currently exist. Toll-like receptors (TLRs) are the major class of DC-activating receptors and have been shown to mediate the effects of all currently used adjuvants. Our proposal is to develop molecules that bind to TLRs with high affinity to simultaneously target and activate DC, thus providing a strong antigen-specific immune stimulus. To identify TLR-binding molecules, single-chain antibody (scFv) libraries will be screened against the purified extracellular domains of all known TLRs. Previous screens by our group have identified large numbers of scFvs that bind to cell surface receptors with high affinity. Such scFvs can be re-engineered as humanized monoclonal antibodies or otherwise manipulated and fused or conjugated to specific antigens. Once identified, candidate scFvs will be tested for their ability to bind TLRs, to bind DC, to target antigens to DC, to activate DC, and to inhibit the activation of DC by natural TLR ligands. (As proposed, our screens are likely to identify scFvs that function as TLR inhibitors.) Those molecules that prove effective in targeting and activating DC will be fused to model antigens derived from anthrax, vaccinia and Yersinia and tested in vivo for their ability to stimulate antibody production, CTL responses, and protective immunity against lethal challenge after systemic and nasal immunization. The primary aim of the proposal is to develop single chain antibodies that: 1) bind specifically to individual toll-like receptors; 2) can be complexed to antigens; 3) target Ag to Ag-presenting cells; 4) activate Ag-presenting cells; 5) stimulate strong Ag-specific immune responses; and 6) provide protective immunity to toxin or pathogen challenge. Once validated, such molecules should be generally applicable as an adjuvant platform by their conjugation or fusion to any protein Ag.